Vaccine against microbial pathogens

ABSTRACT

The present invention provides a vaccine comprising a microbial pathogen, wherein the microbial pathogen is subjected to a stress inducing stimuli. The stress inducing stimuli can be heat or osmotic stress, through preferably the microbial pathogen is genetically modified such that at least one repressor gene for a heat shock protein gene is inactivated, thus allowing the constitutive expression of heat shock proteins. In particular the use of heat shock protein repressor mutant bacteria is shown to be effective for inducing immunity when comprised within vaccines of the present invention. The present invention further provides a method for producing a vaccine comprising stressed induced microbial pathogens and further the use of the heat shock protein repressor deletion mutant microbes as vaccine vectors which can be additionally allow the expression of heterologous antigen fragments.

The present invention relates to a vaccine and a method for producing avaccine. More specifically, there is provided a vaccine comprising amicrobial pathogen and a method of producing the same.

BACKGROUND OF THE INVENTION

An important component of any human immune response is the presentationof antigens to T cells by antigen presenting cells (APCs) such asmacrophages, B cells or dendritic cells. Peptide fragments of foreignantigens are presented on the surface of the macrophage in combinationwith major histocompatibility complex (MHC) molecules, in associationwith helper molecules, such as CD4 and CD8 molecules. Such antigenicpeptide fragments presented in this way are recognised by the T cellreceptor of T cells. The interaction of the antigenic peptide fragmentswith the T cell receptor results in antigen-specific T cellproliferation, and secretion of lymphokines by the T-cells. The natureof the antigenic peptide fragment presented by the APCs is critical inestablishing immunity.

Heat shock proteins (hsps) form a family of highly conserved proteinsthat are widely distributed throughout the plant and animal kingdoms. Onthe basis of their molecular weights, hsps are grouped into sixdifferent families: small (hsp 20-30 kDa); hsp40; hsp60; hsp70; hsp90;and hsp100. Although hsps were originally identified in cells subjectedto heat stress, they have been found to be associated with many otherforms of stress, such as infections, and are thus also commonly referredto as stress proteins (SPs).

Members of the mammalian hsp90 family include cytosolic hsp90 (hsp83)and the endoplasmic reticulum counterparts hsp90 (hsp83), hsp87, Grp94(Erp99) and gp97, see for example Gething et al. (1992) Nature355:33-45. Members of the hsp70 family include cytosolic hsp70 (p73) andhsp70 (p72), the endoplasmic reticulum counterpart BiP (Grp78), and themitochondrial counterpart hsp70 (Grp75). Members of the mammalian hsp60family have only been identified in the mitochondria.

Stress proteins are ubiquitously expressed within cells. One of theroles of stress proteins is to chaperone peptides from one cellularcompartment to another and to present peptides to the MHC molecules forcell surface presentation to the immune system. In the case of diseasedcells, stress proteins also chaperone viral or tumour-associatedpeptides to the cell-surface, see Li and Sirivastave (1994) BehringInst. Mitt, 94: 37-47 and Suzue et al. (1997) Proc. Natl. Acad. Sci. USA94: 13146-51.

The chaperone function of stress proteins is accomplished through theformation of complexes between stress proteins and antigenic peptidefragments, and between stress proteins and viral or tumour-associatedpeptide fragments, in an ATP dependent reaction. The peptide fragmentscomplexed with the stress proteins form antigenic complexes (hspCs)which are captured by APCs to provide antigenic peptide fragments.

The complex association of stress proteins with peptide fragments hasalso been observed in normal tissues and is not therefore atumour-specific phenomenon, see Srivastava (1994) Experimentia 50:1054-60. It is thought that the immunogenicity of the members of thehsp70 family, including grp96, reflects their normal role in the‘presentosome’, the postulated intracellular organelle functioning inthe loading of MHC Class I molecules required for their cell surfaceexpression. This step is essential for their MHC-restricted recognitionby antigen-specific T-cells, see Srivastava et al. (1998) Immunity,Singh-Jasuja et al. J. Exp. Med (2000) 191, 1957.

Until recently, it has not been proposed to use pathogen-derivedendogenous hsp-peptide complexes (hspCs) as vaccines, despite the factthat hsps from pathogens have been used extensively as antigens andadjuvants. PCT Application No GB00/03228 discloses the use ofpathogen-derived endogeneous hspCs and in particular stress inducedhspCs are shown to give good protective immunity in vaccinated animals.

Members of the microbial hsp family include the Dna J and Dna K familiesand the Gro-EL and Gro-ES families. In prokaryotic microbes it appearsthat these families are encoded in operons, with the initial gene in theoperon being a control gene which suppresses the expression of the hspgenes contained within the operon.

In Streptomyces and Helicobacter, expression of the hspR gene suppressesthe expression of Dna J and Dna K. Deletion of the hspR gene thereforeresults in a genetically modified microbe that constitutively expresseshsps, see Bucca et al. (1997) Mol. Microbiol 15:633-45. Homologousoperons have been identified in a number of recently sequenced microbes,including other strains of Streptomyces and Mycobacterium tuberculosisand the commonly used related vaccine strain BCG.

Other repressor genes may also control the expression of members of thehsp gene family and that these may also be genetically engineered toprovide modified microbes that constitutively expresses hspCs that maybe utilised for the production of vaccines and vaccine vectors. Theseinclude, but are not limited to the transcriptional control genes sigmaand rho and the stress-gene regulatory protein genes MerR and HmrR. Itwill also be appreciated that the modified microbes containing theconstitutive hspCs may be used directly as vaccines or as a source forthe isolation of the hspCs. Furthermore the expression of heterologousgene(s) from other pathogen(s) in these microbes will enable their useas vaccine vectors for the simplified production of hspC-based vaccinesfor these pathogens.

Whilst looking at the use of heat-induced endogeneous microbialhsp-peptide complexes, the extracted hsp-peptide complexes were comparedto the use of the stressed-induced microbe itself as a vaccine.Surprisingly, the use of the microbe as a vaccine gave significantlybetter immunity in vaccinated animals than the use of the isolatedhsp-peptide complexes. Similar results were obtained using geneticallymodified microbes that constitutively produced hsps, indicating thathspCs were formed in situ with endogenous microbial polypeptides,including heterogenous genes expressed as recombinant proteins in themicrobe. The genetically modified microbes could also be used as anefficient source for the isolation of hspCs for use in subunit andmulti-subunit vaccines.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a vaccinecomprising a microbial pathogen as an immunogenic determinant, whereinthe microbial pathogen has been subjected to a stress inducing stimuli.

Preferably the stress inducing stimuli results in the expression of heatshock proteins by the microbe.

Preferably the stress inducing stimuli is heat or osmotic shock.

More preferably the stress inducing stimuli is the genetic modificationof the microbial pathogen such that at least one repressor gene for aheat shock protein gene is inactivated, thereby allowing the expressionof a heat shock protein gene.

Preferably the microbial pathogen is any pathogen which is capable ofinducing an infectious disease.

Preferably the microbial pathogen is a bacteria, protozoa, fungi or aparasitic organism.

Preferably the genetic modification results in the inactivation of thehspR repressor gene.

Alternatively the genetic modification results in the inactivation ofthe stress gene regulatory protein genes MerR or HmrR repressor genes,or the transcriptional control genes sigma and rho.

Preferably the microbial pathogen is selected from the group consistingof Mycobacteria, Salmonella, Vibrio, Streptomyces, Helicobacter,Lactococcus and Listeria.

Preferably the microbial pathogen is attenuated.

Preferably the vaccine further comprises an adjuvant.

Preferably the adjuvant is selected from the group consisting of;Freund's complete adjuvant, Freund's incomplete adjuvant, Quil A, Detox,ISCOMs and squalene.

Preferably the vaccine is suitable for administration by injection.

Alternatively the vaccine is suitable for oral administration.

Preferably the vaccine is suitable for delivery by means of aneedle-less delivery format.

A further aspect of the present invention provides a method ofvaccinating an animal, characterised in that said method comprisesadministering a pharmaceutically acceptable quantity of a vaccinecomposition according to the present invention sufficient to elicit animmune response in the animal.

Preferably the vaccine is administered as a prophylactic vaccine.

Alternatively the vaccine is administered as a therapeutic vaccine.

Preferably the vaccine composition is administered by injection.

Alternatively the vaccine composition is administered by needle-lessdelivery.

Alternatively the vaccine composition is administered transdermally, bypulmonary delivery or orally.

A yet further aspect of the present invention provides a method ofproducing a vaccine composition, comprising an immunogenic determinant,characterised in that said method comprises the steps of; subjectingmicrobial pathogens to stress inducing stimuli; and using the stressedmicrobe in the preparation of the vaccine composition as said imunogenicdeterminant.

Preferably the stress inducing stimuli is heat or osmotic shock.

More preferably the stress inducing stimuli is the inactivation of agene which represses the expression of heat shock genes.

Preferably the repressed gene is the hspR gene.

Alternatively the repressed gene is the stress gene regulatory proteingene MerR or HmrR, or the transcriptional control genes sigma and rho.

Preferably the microbial pathogen cells are killed prior to use in saidvaccine.

Alternatively the microbial pathogen cells are dried prior to use insaid vaccine.

Preferably the vaccine composition is an aqueous composition.

Alternatively the vaccine composition is a dry composition or in alyophilised composition.

The present invention further provides the use of a pathogenic microbewhich has been genetically modified such that at least one repressorgene for a heat shock protein is inactivated, thereby allowingconstitutive expression of heat shock proteins, as a vaccine vectorwhich further allows the expression of a heterologous antigen.

Preferably the pathogenic microbe is a prokaryotic organism, which issuitable for use as a vaccine vector.

Preferably the prokaryotic organism is BCG and the heterologous antigenfragment is the tetanus toxoid fragment C.

Alternatively the prokaryotic organism is Salmonella or Lactococcus,such that the vaccine can be administered orally.

The present invention further provides the use of a pathogenic microbewhich has been genetically modified such that at least one repressorgene for a heat shock protein is inactivated, thereby allowingconstitutive expression of heat shock proteins, as a source of heatshock protein-peptide complexes for use in subunit and multi-subunitvaccines.

By “microbial pathogen” it is meant any pathogen capable of inducing aninfectious disease in an animal, including particularly bacterial,protozoan, fungal or parasitic organisms.

By “stress-inducing stimuli” it is meant any stimuli that induces theproduction of SPs and in particular hsps in microbial pathogens,including heat or osmotic stress. Such stress-inducing stimuli alsoincludes genetic changes designed to render constitutive the expressionof stress proteins and in particular hsps in microbial pathogens. Thesegenetic changes include the inactivation of repressor (suppressor) genesthat function in the suppression of stress proteins and in particularthe suppression of the genes which express heat shock proteins. Thisincludes in particular the inactivation of the hspR suppressor gene, thestress gene regulatory protein genes MerR and HmrR, or thetranscriptional control genes sigma and rho.

It will be appreciated that such genetic changes are readily achieved bymolecular genetic means and include insertional mutagenesis using phageor transposon vectors, see for example Bucca et al. Mol. Microbiol.(1997) 17:633. It will also be appreciated that the presence of thegenes for the operons, suppressor genes and SPs and hsps in microbialpathogens can be simply established by recombinant DNA techniques, seeCurrent Techniques in Molecular Biology, (1999) Wiley Press.

Thus it should be appreciated that any microbe expressing these genescan be genetically modified to provide a vaccine according to thisinvention.

This includes existing microbial organisms used as live or killedvaccines, such as Mycobacteria, Salmonella, Vibrio and Listeria and inparticular attenuated varieties of these pathogens. In particular,Mycobacteria mutants, which have been genetically modified to delete theheat shock protein repressor gene could be used in a vaccine againsttuberculosis, while Salmonella mutants which have been geneticallymodified to delete the heat shock protein repressor genes could be usedin a vaccine against typhoid. The use of such organisms as vaccinevectors for the expression of heterologous genes is further provided bythe present invention.

The term “vaccine” is used herein to denote to any compositioncontaining an immunogenic determinant which stimulates the immune systemsuch that it can better respond to subsequent infections. It will beappreciated that a vaccine usually contains an immunogenic determinantand an adjuvant, the adjuvant serving to non-specifically enhance theimmune response to that immunogenic determinant.

Suitable adjuvants are readily apparent to the person skilled in theart, and include; Freund's complete adjuvant, Freund's incompleteadjuvant, Quil A, Detox, ISCOMs or squalene.

However, it will be appreciated that the vaccine of the presentinvention may also be effective without an adjuvant.

For the non-genetic induction of SPs, the optimum conditions forinducing the SPs can readily be determined by simple trial and errorwith the effect of a change of stimuli being assessed using conventionaltechniques, such as in vivo testing on animals, or by other techniques,for example those described in ‘Current Protocols in Immunology’, WileyInterscience, 1997. Other such conditions are described in PCTApplication No GB00/03228 and citations referred to therein.

The invention also provides a method for exposing an animal to a vaccineof the invention by administering a pharmaceutically acceptable quantityof the vaccine of the invention, optionally in combination with anadjuvant, sufficient to elicit an immune response in the animal.

The animal is typically a human. However, the invention can also beapplied to the treatment of other mammals such as horses, cattle, goats,sheep or swine, and to the treatment of birds, notably poultry such aschicken or turkeys. Preferably the microbial pathogen selected for usein a particular vaccine of the present invention causes disease orinfection in the species of animal to which the vaccine is administeredto, or a closely related species. The vaccines of this invention may beused as both prophylactic or therapeutic vaccines though it will beappreciated that they will be particularly useful as prophylacticvaccines due to their economy of production.

The vaccine compositions of the present invention may be administered byany suitable means, such as orally, by inhalation, transdermally or byinjection and in any suitable carrier medium. However, it is preferredto administer the vaccine as an aqueous composition by injection usingany suitable needle or needle-less technique.

It will be appreciated that the vaccine of the invention may be appliedas an initial treatment followed by one or more subsequent “booster”treatments at the same or a different dosage rate at an interval of from1 to 26 weeks between each treatment to provide prolonged immunisationagainst the pathogen.

The present invention will now be described, by way of example, withreference to the accompanying figures, wherein;

FIG. 1 shows the nucleotide sequence of the hspR gene of M.tuberculosis, and

FIG. 2 shows a list of identified suppressor genes which have homologyto hspR.

EXAMPLE 1 Preparation of Heat-Induced Microbes

M. vaccae strain NCTC11659 was grown to saturation in Sauton's media,diluted into fresh media and grown overnight to give a log phase culturewhich was then heat-shocked at 42° C. for 3 hours or at 39° C. for 5hours and cultured overnight. The cells were then washed in media,followed by a wash in saline and either lyophilised in individualvaccine aliquots or used directly for the immunisation of test animals.For comparison isolated endogeneous SP-peptide complexes were preparedas described in PCT Application No GB00/03228. Essentially, washedstress-induced cells are then re-suspended in homogenisation buffer suchas PBS with 0.5% Tween and cells are then disrupted by freeze-thawcycles or using cell disruptor (e.g. bead beater, French press). Thecell lysate is then treated by centrifugation, typically 3-5000 g for 5minutes, to remove the nuclei and cell debris, followed by a high speedcentrifugation step, typically 100,000 g for 15-30 minutes. Thesupernatant thus obtained is processed to give an SP/antigenic peptidefragment complex suitable for use in a vaccine. This can be done simplyby ammonium sulphate precipitation which uses a 20-70% ammonium sulphatecut. Specifically, 20% (w/w) ammonium sulphate is added at 4° C., theprecipitate is discarded, followed by the addition of more ammoniumsulphate to bring the concentration to 70% w/w. The protein precipitateis harvested by centrifugation and then dialysed into an appropriatephysiological, injectable buffer, such as saline, to remove the ammoniumsulphate before use. The SP complexes may be used at any suitableconcentration to provide the immunogenic determinant in the vaccinecomposition.

It is preferred that the amount of the induced stress protein-peptidecomplex is in the range of 10-600 μg, more preferably 10-100 μg, andmost preferably 25 μg per kg of animal body weight.

In order to determine the immunogenicity of the SP complexes, T cellproliferation assays may be used. Suitable assays include themixed-lymphocyte reaction (MLR), assayed by tritiated thymidine uptake,and cytotoxicity assays to determine the release of ⁵¹Cr from targetcells, see ‘Current Protocols in Immunology’, Wiley Interscience, 1997.For Mycobacteria, MLRs can also be assayed for the induction of cytokineproduction such as the production of interferon gamma using thecommercial kit (CSL Ltd). Alternatively, antibody production may beexamined, using standard immunoassays or plaque-lysis assays, orassessed by intrauterine protection of a foetus, see ‘Current Protocolsin Immunology’.

Mice were immunised with either heat-stressed organisms or theendogenous SP-peptide complexes isolated from the heat stressedorganisms in phosphate buffered saline without any added adjuvant ineither the primary or booster vaccinations.

Immunisation with whole stress-induced organisms and in particularlyophilised organisms gave significantly better immunity than thatinduced by isolated endogeneous SP-peptide complexes including increasedIFN-gamma and antibody production.

EXAMPLE 2 Preparation and Use of Constitutive hsp Mutants

Constitutive hsp producing microbes can be constructed through theknockout of transcriptional regulator suppressor genes such as hspR,MerR (mercuric resistance operon regulatory protein), HmrR (heavy metalregulatory protein) and their homologues or the transcriptional controlgenes rho and sigma. Such genes are readily identified by screening ofgenome sequence databases with typical test sequences. An example ofsuch a typical test sequence is the hspR gene of M. tuberculosis (shownin FIG. 1). An example of a list of identified suppressors which havehomology to the hspR gene is shown in FIG. 2.

M. bovis strains constitutively producing hspCs were constructed bygenetically engineering the M. bovis strain BCG to yield deletionmutants for the hspR gene. The hspR gene was deleted by homologousrecombination using a suicide vector carrying a large fragment of thehspR gene and a kanamycin selection marker. The hspR gene fragment wascloned by PCR from BCG genomic DNA using primers derived from the M.tuberculosis hspR sequence shown in FIG. 1. Such an approach should bewidely applicable to the production of similar mutants from anyprokaryote carrying the relevant suppressor gene.

BCG hspR deletion mutants were used to produce hspCs and the proteinyields obtained from these were comparable to those obtained from heatshocked wild type strains. Such an approach should again be widelyapplicable to the production of hspCs from any prokaryote carrying therelevant suppressor gene. These would provide a valuable resource forthe manufacture of hspC-based vaccines. Thus hspCs produced from BCGhspR deletion mutants induced protective immunity against aerosolchallenge by M. tuberculosis comparable to that induced by hspCsobtained from heat shocked wild type BCG strains.

BCG hspR deletion mutants can be also be used as vaccine vectors toexpress heterologous antigens. For example, mice immunised with a BCGhspR deletion mutant expressing the tetanus toxoid (TT) fragment Cshowed the production of anti-TT antibodies as detected by Westernblotting. Such an approach should be widely applicable to the productionof similar vectors for the expression of heterologous vaccine antigensusing mutants from any prokaryote suitable for use as a vaccine vector.For example the use of Salmonella or Lactococcus mutants would enablethe production of vectors targeted for mucosal delivery of vaccines.Vaccines developed on this principle would be particularly advantageousin that they could be administered orally.

1. A vaccine comprising a microbial pathogen as an immunogenic determinant, wherein the microbial pathogen has been subjected to a stress inducing stimuli.
 2. A vaccine as claimed in claim 1 wherein the stress inducing stimuli results in the expression of heat shock proteins by the microbe.
 3. A vaccine as claimed in claim 1 or claim 2, wherein the stress inducing stimuli is heat or osmotic shock.
 4. A vaccine as claimed in claims 1 to 3, wherein the stress inducing stimuli is the genetic modification of the microbial pathogen such that at least one repressor gene for a heat shock protein gene is inactivated, thereby allowing the expression of a heat shock protein gene.
 5. A vaccine as claimed in any of the preceding claims, wherein the microbial pathogen is any pathogen which is capable of inducing an infectious disease.
 6. A vaccine as claimed in any of the preceding claims, wherein the microbial pathogen is a bacteria, protozoa, fungi or a parasitic organism.
 7. A vaccine as claimed in claims 4 to 6, wherein the genetic modification results in the inactivation of the hspR repressor gene.
 8. A vaccine as claimed in claims 4 to 6, wherein the genetic modification results in the inactivation of the stress gene regulatory protein genes MerR or HmrR.
 9. A vaccine as claimed in claims 4 to 6, wherein the genetic modification results in the inactivation of the transcription control genes rho or sigma.
 10. A vaccine as claimed in any of the preceding claims wherein the microbial pathogen is selected from the group consisting of Mycobacteria, Salmonella, Vibrio, Listeria, Streptomyces, Helicobacter and Lactococcus.
 11. A vaccine as claimed in any of the preceding claims wherein the microbial pathogen is attenuated.
 12. A vaccine as claimed in any of the preceding claims, wherein the vaccine further comprises an adjuvant.
 13. A vaccine as claimed in claim 12, wherein the adjuvant is selected from the group consisting of; Freund's complete adjuvant, Freund's incomplete adjuvant, Quil A, Detox, ISCOMs and squalene.
 14. A vaccine as claimed in any of the preceding claims, wherein the vaccine is suitable for administration by injection.
 15. A vaccine as claimed in claims 1 to 13 wherein the vaccine is suitable for oral administration.
 16. A vaccine as claimed in claim 14, wherein the vaccine is suitable for delivery by means of a needle-less delivery format.
 17. A method of vaccinating an animal, characterised in that said method comprises administering a pharmaceutically acceptable quantity of a vaccine composition as claimed in any of claims 1 to 13, sufficient to elicit an immune response in the animal.
 18. A method as claimed in claim 17 wherein said vaccine is administered as a prophylactic vaccine.
 19. A method as claimed in claim 17 wherein said vaccine is administered as a therapeutic vaccine.
 20. A method as claimed in claims 17, wherein the vaccine composition is administered by injection.
 21. A method as claimed in claims 17, wherein the vaccine composition is administered by needle-less delivery.
 22. A method as claimed in claims 17, wherein the vaccine composition is administered transdermally.
 23. A method as claimed in claims 17, wherein the vaccine composition is administered by pulmonary delivery.
 24. A method as claimed in claim 17, wherein the vaccine composition is administered orally.
 25. A method of producing a vaccine composition, comprising an immunogenic determinant, characterised in that said method comprises the steps of: subjecting microbial pathogens to stress inducing stimuli; and using the stressed microbe in the preparation of the vaccine composition as said imunogenic determinant.
 26. A method as claimed in claim 24, characterised in that the stress inducing stimuli is heat or osmotic shock.
 27. A method as claimed in claim 25, characterised in that the stress inducing stimuli is the inactivation of a gene which represses the expression of heat-shock genes.
 28. A method as claimed in claim 25, characterised in that the repressor gene is the hspR gene.
 29. A method as claimed in claim 25, characterised in that the repressor gene is MerR or HmrR.
 30. A method as claimed in any one of the preceding claims, characterised in that the microbial pathogen cells are killed prior to use in said vaccine.
 31. A method as claimed in any of the preceding claims, characterised in that the microbial pathogen cells are dried prior to use in said vaccine.
 32. A vaccine composition as claimed in any one of claims 25 to 31, characterised in that the composition is an aqueous composition.
 33. A vaccine composition as claimed in any one of claims 25 to 31, characterised in that the composition is a dry composition.
 34. A vaccine composition as claimed in claims 25 to 31, characterised in that the composition is a lyophilised composition.
 35. Use of a pathogenic microbe which has been genetically modified such that at least one repressor gene for a heat shock protein is inactivated, thereby allowing constitutive expression of heat shock proteins, as a vaccine vector which further allows the expression of a heterologous antigen.
 36. Use of pathogenic microbe as claimed in claim 34 wherein the pathogenic microbe is a prokaryotic organism, which is suitable for use as a vaccine vector.
 37. Use of pathogenic microbe as claimed in claim 35 wherein the prokaryotic organism is BCG and the heterologous antigen fragment is the tetanus toxoid fragment C.
 38. Use of pathogenic microbe as claimed in claim 34 wherein the prokaryotic organism is Salmonella or Lactococcus, such that the vaccine can be administered orally.
 39. Use of a pathogenic microbe which has been genetically modified such that at least one repressor gene for a heat shock protein is inactivated, thereby allowing constitutive expression of heat shock proteins, as a source of heat shock protein-peptide complexes for use in subunit and multi-subunit vaccines. 